Virus Multiplication

Virus Multiplication (Lecture 21)
• Virus – consists of a cluster of genes that can replicate insides cells
• Simplicity in their genetic organization  consist of DNA or RNA, not both
• Obligate intracellular parasites
Patterns of virion structure
• Core – viral nucleic acid
• Protein shell  capsid – either icosahedral or helical
• Some viruses have lipoprotein envelope surrounding the capsid
• Capsid – built from multiple copies of identical structural units
• Icosahedron – 20 triangular faces, 12 vertices  2,3 and 5 fold symmetry
Other virion proteins
• Contain small amounts of proteins that play critical roles after infection
• Sometimes they contain enzymes that catalyze rxns that do not normally occur in
unifected animal cells, such as RNA-dependent RNA polymerase or reverse
transcriptase  good targets for anti-viral therapy since not in animal cells
• Other times, they are integrases, transcription factors, and proteases
Classification
• DNA or RNa, polarity (plus  same as mRNA, or minus), structure (linear,
circular, single- or double- stranded)
• Type of symmetry (helical or icosahedral) of capsid
• Presence/absence of lipoprotein envelope
• Presence/absence of specific virion enzymes
Virus infection
• One-step growth curve
• Eclipse – very few infectious virus particles can be detected either inside or
outside the cell during this time
• Intracellular accumulation period – newly-synthesized progeny virions can be
found inside but not outside the cell
• Rise period – increasing numbers of progeny virions appear in the extracellular
fluid
• 2 ways to count viruses:
o functional – apply virus to layer of cells, count plaques (pfu)
o physical – count viral particles with electron microscope
o particle number is usually greater than pfu number
3 basic processes common to all virus infections:
• Delivery of viral genome into the cell  involves attachment of the virion to the
cell and liberation of the viral nucleic acid
• Synthesis of new viral genomes and virion structural proteins • Assembly and release of infectious progeny virions
Attachment
• Involves interaction between certain proteins on the virion surface and specific
receptors on the cell surface
• Receptors are normal membrane proteins or carbohydrates
• Some receptors found in most cells while others are specific to certain cell types
• Specificity of attachment determines the virus host range
Penetration and uncoating
• Viruses enter the cell by two major routes
• Endocytosis – both enveloped and nonenveloped are taken into cells this way
o following attachment, the virus-receptor complex migrates to coated pits
and enter the cell in clathrin-coated vesicles produced by pinching off of
invaginations of the cytoplasmic membrane
o this is analogous to receptor mediated endocytosis of nonviral ligands
o the acidic pH of the endosome triggers conformational changes that
activate the fusion proteins of enveloped viruses  the resulting fusion of
the viral envelope with the membrane of the endosome releases the viral
nucleocapsid into the cytoplasm
o uncertain how nonenveloped viruses escape from the endosome
o influenza virus HA protein  sheds light on mechanism of acid-induced
membrane fusion
 acidic pH causes a loop segment in the HA protein to adopt an
alpha helical conformation
 the result of this is that a hydrophobic segment (fusogenic peptide)
at the N-terminus of ha2 is moved over 100 angstroms from a
buries position to the top of the HA molecules where is inserts into
the membrane of the endosome.
• Direct fusion
o Paramyxoviruses (and a few others) enter cells by fusion of the viral
envelope membrane with the cytoplasmic membrane
o Process requires a specific fusion protein (F protein ) on the viral
envelope.
o Membrane fusion results in the delivery of a naked nucelopcapsid to
cytoplasm of the cell – viral envelope glycoproteins are left embedded in
the plasma membrane
Synthetic phase of virus multiplication
SV40
• naked icosahedral capsid surrounding the viral genome which is complexed with
histones. Genome is dsDNA ~5000 bp (one of the smaller virus genomes)
• 4 genes essential for virus multiplication (T antigen, VP1, VP2, VP3) • SV40 virions enter cell by endocytosis  after uncoating, the viral genome
migrates into cell nucleus when multiplication takes place
• 2 phases of viral genome expression
o Early phase (prior to viral DNA replication) – the early region of viral
genome transcribed into mRNA which directs synthesis of the T antigen
(aka A protein)
 T antigen accumulates in the nucleus where it performs several
important functions
• Induces the host cell to enter S phase so that cellular
enzymes involved in DNA replication are expressed (e.g
DNA pol, ligase, nucleoside kinases)
• Initiates the SV40 DNA replication at the viral origin
(serves as helicase)
• Activates late mRNA synthesis (boundary between early
and late phases)
• Represses early mRNA synthesis (own synthesis)
• Prevents the host cell from undergoing apoptosis
 T antigen – site-specific DNA binding protein with intrinsic
helicase activity
• Initiation of SV40 DNA replication occurs when T antigen
binds to the origin of replication and unwinds the 2 parental
strands of DNA. It also recruits cellular replication
proteins to the origin resulting the in the establishment of 2
replication forks. The forks proceed bidirectionally from
the origin, syntheisizing in the semi-conservative
mechanism. 2 daighter molecules are produced when the
growing points meet roughly 180 degrees around the
genome.
o Late phase – at this time mRNAs transcribed from the late region of the
genome appear in the cytoplasm and direct the synthesis of VP1, VP2, and
VP3. These mRNAs synt